基于MAPK通路探讨消痈止痢汤对溃疡性结肠炎大鼠的抗炎机制

Exploration on the anti-inflammatory mechanism of Xiaoyongzhili decoction on ulcerative colitis rats based on MAPK pathway

目的 基于丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路,探究消痈止痢汤对葡聚糖硫酸钠(Dextran Sulphate Sodium Salt,DSS)诱导溃疡性结肠炎(ulcerative colitis,UC)大鼠的治疗作用及机制。方法 将51只SPF级SD大鼠适应性喂养7天后,随机分为正常组和造模组。对造模大鼠自由饮用4.0%DSS溶液,7天建立UC大鼠模型,分为模型组、消痈止痢汤低、中、高剂量组和柳氮磺吡啶组,连续7天灌胃治疗。实验期间,每日记录大鼠的体质量、大便性状及隐血情况,疗程后取材,测量结肠长度、计算疾病活动指数(Disease activity index,DAI)评分,苏木素-伊红(HE)染色后观察组织形态,酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)法检测血清中白介素(interleukin,IL)-1β、IL-6、肿瘤坏死因子(tumor necrosis factor,TNF)-α、髓过氧化物酶(myeloperoxidase,MPO)含量,免疫蛋白印迹(Western blot)法对组织中细胞外信号调节激酶(Extracellular Signal-regulated Kinases,ERK)、c-Jun氨基末端激酶(c-Jun N-terminal Kinases,JNK)、p38MAPK、核因子-κBp65(Nuclear Factor Kappa-B,NF-κBp65)蛋白及其磷酸化(phosphorylation,p)蛋白进行检测,免疫组化法对结肠组织中磷酸化ERK、JNK、p38MAPK、NF-κBp65蛋白进行检测。结果 与正常组比较,模型组结肠长度缩短,DAI评分增高,黏膜破损明显,血清中IL-1β、IL-6、TNF-α、MPO含量增加,组织中ERK、JNK、p38MAPK、NF-κBp65蛋白及磷酸化蛋白含量均增加(P<0.01);与模型组比较,各剂量中药组及西药组结肠长度增加,DAI评分降低,血清中IL-1β、IL-6、TNF-α、MPO含量降低(P<0.05,P<0.01);Western blot结果显示:与模型组比较,除了低剂量组在p-JNK蛋白含量上无明显差异外(P>0.05),各不同剂量中药组及西药组组织中ERK、JNK、p38MAPK、NF-κBp65及p-ERK、p-JNK、p-p38MAPK、p-NF-κBp65蛋白水平显著降低(P<0.05,P<0.01);免疫组化结果显示:与模型组比较,消痈止痢汤中、高剂量组和西药组能下调组织中p-ERK、p-JNK、p-p38MAPK、p-NF-κBp65蛋白表达(P<0.05,P<0.01),低剂量组能下调p-ERK、p-NF-κBp65蛋白表达(P<0.05,P<0.01)。结论 消痈止痢汤对DSS诱导UC大鼠有显著治疗作用,其机制可能与抑制MAPK信号通路活化,降低促炎性细胞因子分泌与合成,抑制免疫及炎症反应有关。

Objective Based on mitogen activated protein kinase(MAPK) signaling pathway,to investigate the effect and mechanism of Xiaoyongzhili decoction on sodium glucan sulfate(DSS) induced ulcerative colitis(UC) rats.Methods After adaptive feeding for 7 days,51 SPF SD rats were randomly divided into blank group and model group.The model rats were free to drink 4.0%DSS solution,and UC rat model was established for 7 days.The rats were divided into model group,Xiaoyongzhili decoction low-dose,medium-dose and high-dose groups and sulfamyridine salazide group.During the experiment,the body weight,fecal traits and occult blood of the rats were recorded daily.After treatment,the rats were sampled,the length of colon was measured,the disease activity index(DAI) score was calculated,and the tissue morphology was observed after hematoxylin-eosin(HE) staining.The contents of interleukin-1β(IL-1β),IL-6,tumor necrosis factor-α(TNF-α) and myeloperoxidase(MPO) in serum were determined by enzyme-linked immunosorbent assay(ELISA).Extracellular signal-regulated kinase(ERK),C-Jun amino-terminal kinase(JNK),p38 MAPK,nuclear factor-κBp65(NF-κBp65) protein and its phosphorylation(P) protein were detected by Western blot.The phosphorylated ERK,JNK,p38 MAPK and NF-κBp65 proteins were detected by immunohistochemistry.Results Compared with normal group,colon length of model group was shortened,DAI score was increased,mucosal damage was obvious,serum IL-1β,IL-6,TNF-α and MPO contents were increased.The protein contents and the phosphorylated protein of ERK,JNK,p38 MAPK,NF-κBp65 and p-ERK,p-JNK,p-P38 MAPK,p-NF-κBp65 were increased in tissues(P< 0.01).Compared with model group,colon length increased,DAI score decreased,serum IL-1β,IL-6,TNF-αand MPO contents decreased in TCM and western medicine groups.Western blot results showed that: Compared with model group,there was no significant difference in p-JNK protein content in low dose group(P > 0.05).The protein levels of ERK,JNK,p38 MAPK,NF-κBp65 and p-ERK,p-JNK,p-P38 MAPK and p-NF-κBp65 in different doses of Traditional Chinese medicine and western medicine groups were significantly decreased(P< 0.05,P < 0.01).Immunohistochemical results showed that: Compared with model group,the protein expression of p-ERK,p-JNK,p-P38 MAPK and p-NF-κBp65 in Xiaoyongzhili decoction medium and high dose groups and western medicine group could be down-regulated(P < 0.05,P < 0.01),and the protein expression of p-ERK and p-NF-κBp65 in low dose group could be down-regulated(P < 0.05,P < 0.01).Conclusion Xiaoyongzhili decoction has significant therapeutic effect on DSS induced UC rats,and its mechanism may be related to inhibiting the activation of MAPK signaling pathway,reducing the secretion and synthesis of pro-inflammatory cytokines,and inhibiting immune and inflammatory responses.